Probiotic‑derived silver nanoparticles target mTOR/MMP‑9/BCL‑2/dependent AMPK activation for hepatic cancer treatment

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Medical Oncology (2024) 41:106
https://doi.org/10.1007/s12032-024-02330-8
ORIGINAL PAPER
Probiotic‑derived silver nanoparticles target mTOR/MMP‑9/BCL‑2/
dependent AMPK activation forhepatic cancer treatment
AlaaElmetwalli1,2 · MohamedO.Abdel‑Monem3· AliH.El‑Far4· GehadS.Ghaith3· NoafAbdullahN.Albalawi5·
JihanHassan6· NadiaF.Ismail7· TarekEl‑Sewedy6· MashaelMashalAlnamshan8· NoufK.ALaqeel8·
IbtesamS.Al‑Dhuayan8· MervatG.Hassan3
Received: 7 January 2024 / Accepted: 8 February 2024
© The Author(s) 2024
Abstract
Recent advances in nanotechnology have offered novel ways to combat cancer. By utilizing the reducing capabilities of
Lactobacillus acidophilus, silver nanoparticles (AgNPs) are synthesized. The anti-cancer properties of AgNPs have been
demonstrated in previous studies against several cancer cell lines; it has been hypothesized that these compounds might
inhibit AMPK/mTOR signalling and BCL-2 expression. Consequently, the current research used both invitro and in silico
approaches to study whether Lactobacillus acidophilus AgNPs could inhibit cell proliferation autophagy and promote
apoptosis in HepG2 cells.The isolated strain was identified as Lactobacillus acidophilus strain RBIM based on 16s rRNA
gene analysis. Based on our research findings, it has been observed that this particular strain can generate increased quanti-
ties of AgNPs when subjected to optimal growing conditions. The presence of silanols, carboxylates, phosphonates, and
siloxanes on the surface of AgNPs was confirmed using FTIR analysis. AgNPs were configured using UV–visible spectros-
copy at 425nm. In contrast, it was observed that apoptotic cells exhibited orange-coloured bodies due to cellular shrinkage
and blebbing initiated by AgNP treatment, compared to non-apoptotic cells. It is worth mentioning that AgNPs exhibited
remarkable selectivity in inducing cell death, specifically in HepG2 cells, unlike normal WI-38 cells. The half-maximum
inhibitory concentration (IC50) values for HepG2 and WI-38 cells were 4.217µg/ml and 154.1µg/ml, respectively. AgNPs
induce an upregulation in the synthesis of inflammation-associated cytokines, including (TNF-α and IL-33), within HepG2
cells. AgNPs co-treatment led to higher glutathione levels and activating pro-autophagic genes such as AMPK.
Additionally, it resulted in the suppression of mTOR, MMP-9, BCL-2, and α-SMA gene expression. The docking experiments
suggest that the binding of AgNPs to the active site of the AMPK enzyme leads to inhibiting its activity. The inhibition of
AMPK ultimately results in the suppression of the mechanistic mTOR and triggers apoptosis in HepG2 cells. In conclusion,
the results of our study indicate that the utilization of AgNPs may represent a viable strategy for the eradication of liver
cancerous cells through the activation of apoptosis and the enhancement of immune system reactions.
Keywords AgNPs· Apoptosis· Hepatic cancer· Lactobacillus acidophilus· α-SMA· HepG2
* Alaa Elmetwalli
dr.prof2011@gmail.com
1 Department ofClinical Trial Research Unit andDrug
Discovery, Egyptian Liver Research Institute andHospital
(ELRIAH), Mansoura, Egypt
2 Microbiology Division, Higher Technological Institute
ofApplied Health Sciences, Egyptian Liver Research
Institute andHospital (ELRIAH), Mansoura, Egypt
3 Botany andMicrobiology Department, Faculty ofScience,
Benha University, Benha, Egypt
4 Department ofBiochemistry, Faculty ofVeterinary
Medicine, Damanhour University, Damanhour22511, Egypt
5 Department ofNursing, Al Hadithah General Hospital,
AlQurayyat, SaudiArabia
6 Department ofApplied Medical Chemistry, Medical
Research Institute, Alexandria University, Alexandria, Egypt
7 Health Information Management Program, Biochemistry,
Faculty ofHealth Science Technology, Borg El Arab
Technological University, Alexandria, Egypt
8 Biology Department, College ofScience, Imam
Abdulrahman Bin Faisal University, 31441Dammam,
SaudiArabia

Medical Oncology (2024) 41:106 106 Page 2 of 20
Introduction
Cancer is a global health issue affecting millions of people
worldwide. Liver cancer, in particular, has become a sig-
nificant concern due to its high mortality rate [1, 2]. Tra-
ditional cancer treatment methods often come with severe
side effects, highlighting the need for alternative therapeu-
tic approaches [3–5]. In recent years, nanotechnology has
emerged as a promising field in cancer research, offering
novel ways to combat this devastating disease [6]. The
synthesis of silver nanoparticles (AgNPs) from Lactoba-
cillus acidophilus involves a bio-reduction approach that
utilizes the reducing properties of the bacterial cells [7].
In bacteria-synthesized AgNPs, the arrangement of
Ag atoms varies significantly from species to species [8].
Those differences can be attributed to the fact that different
microorganisms have different enzymes that are involved
in the reduction of Ag-oxyanions. These enzymes can
modify the structure, size, and shape of AgNPs and affect
their properties [9]. Furthermore, the bacterial species can
also influence the environment in which the AgNPs are
produced, thus further influencing their characteristics
[10]. Among these bacteria is Lactobacillus acidophilus,
a probiotic bacterium that has gained significant atten-
tion for its potential anti-cancer properties [11]. Research-
ers have utilized Lactobacillus acidophilus to synthesize
AgNPs due to its ability to reduce silver ions into their
nanoparticle form [7, 12]. The synthesis process involves
the use of Lactobacillus acidophilus culture filtrate, which
acts as a reducing and stabilizing agent for the formation
of AgNPs. The resulting nanoparticles exhibit unique
physicochemical properties that make them suitable for
various biomedical applications, including cancer therapy
[13].
The pharmaceutical and biological potential of green-
synthesized AgNPs has gained attention due to their
unique properties [14]. The following are some of their
effects; an array of viruses are susceptible to the antiviral
agents in AgNPs, including influenza and herpes simplex
virus. Their function is to inhibit viral attachment and
entry into host cells. In addition, they inhibit viral pro-
tein synthesis, making them potentially effective against
viruses [15]. A biofilm is a community of microorganisms
encased in an extracellular polymer matrix that resists
antibiotics. Biofilms are disrupted, microbial adhesion is
inhibited, and embedded bacteria are killed by AgNPs. As
a result, they can prevent biofilm-associated infections,
such as those associated with medical devices [16].
Analgesic effects of AgNPs have been explored. They
reduce oxidative stress and inhibit pro-inflammatory medi-
ators, modulating inflammatory responses. In addition,
AgNPs can enhance the effectiveness of conventional pain
medications [17]. The efficacy and safety of these drugs
must be validated in clinical studies. The anticoagulant
properties of AgNPs are thought to arise from the interac-
tion of AgNPs with blood components. Thrombus forma-
tion can be prevented by inhibiting platelet aggregation
and adhesion to vascular surfaces. Additionally, AgNPs
interfere with factors involved in blood clotting, modulat-
ing the coagulation cascade [18].
AgNPs possess unique properties due to their small size
and large surface area-to-volume ratio. Their nanoscale
dimensions allow for better penetration into cancer cells,
enhancing their therapeutic potential [19]. The increased
surface area enables efficient interaction with cellular com-
ponents, aiding in their anti-cancer activity. AgNPs have
been shown to be readily taken up by liver cancer cells.
Once inside the cells, they can interact with various cellular
targets, triggering apoptotic pathways. This cellular uptake
is facilitated by the nanoparticles' small size and surface
modifications, which can enhance their internalization [20].
AgNPs have been reported to induce mitochondrial dys-
function in liver cancer cells, further contributing to their
apoptotic effects [21, 22]. These AgNPs can disrupt the
mitochondrial membrane potential, impairing energy pro-
duction and triggering apoptotic signalling pathways. This
disruption of mitochondrial function is considered a crucial
step in the induction of apoptosis [23].
Apoptosis, or programmed cell death, is a tightly regu-
lated process responsible for maintaining tissue homeostasis.
Dysregulation of apoptosis can contribute to cancer develop-
ment and progression [24–26]. AgNPs derived from Lacto-
bacillus acidophilus have been found to induce apoptotic
cell death in liver cancer cells [27]. They activate various
signalling pathways and promote the release of pro-apoptotic
proteins, leading to the fragmentation and subsequent death
of cancer cells. This apoptotic cell death induction offers
a potential therapeutic strategy for liver cancer treatment
[28, 29].
The AMP-activated protein kinase (AMPK) and mam-
malian target of rapamycin (mTOR) signalling pathway
is a crucial cellular pathway that regulates various cellu-
lar processes, including autophagy [30, 31]. Under normal
conditions, mTOR inhibits autophagy by phosphorylating
and inactivating autophagy-related proteins [32]. However,
in the presence of cellular stress, such as nutrient depriva-
tion or oxidative stress, AMPK is activated, leading to the
inhibition of mTOR and subsequent induction of autophagy
[33]. Matrix metalloproteinase-9 (MMP-9) is an enzyme that
plays a crucial role in extracellular matrix remodelling and
cell migration [34]. In liver cancer cells, MMP-9 has been
found to interact with B-cell lymphoma 2 (BCL-2) [35], an
anti-apoptotic protein that prevents cell death. The interac-
tion between MMP-9 and BCL-2 can promote cell survival
and contribute to cancer progression [36].

Medical Oncology (2024) 41:106 Page 3 of 20 106
AgNPs have been shown to activate the AMPK/mTOR
signalling pathway, leading to autophagy induction in liver
cancer cells. AgNPs exert their effect by modulating the
phosphorylation status of AMPK and mTOR [37]. Stud-
ies have demonstrated that AgNPs can increase AMPK
phosphorylation and inhibit mTOR signalling, resulting in
the induction of autophagy [38, 39]. This activation of the
AMPK/mTOR pathway by AgNPs promotes the degrada-
tion of damaged proteins and organelles, ultimately leading
to cell death in liver cancer cells [40]. Furthermore, AgNPs
have also been found to regulate the MMP-9-BCL-2 sig-
nalling pathway [41]. Studies have shown that AgNPs can
inhibit MMP-9 activity, thereby reducing its interaction with
BCL-2 [42, 43]. This disruption of the MMP-9-BCL-2 inter-
action leads to decreased cell survival and increased suscep-
tibility to apoptosis, further contributing to the anti-cancer
effect of AgNPs in liver cancer cells [44].
As part of this study, we synthesized biogenic AgNPs
using Lactobacillus acidophilus. Furthermore, Lactoba-
cillus acidophilus-enriched nano-Ag was evaluated for its
efficacy in suppressing liver cancer cells as dietary supple-
ments/nutraceuticals. AgNPs were also investigated for their
ability to induce apoptosis and contribute to the immune
response against liver cancer cells. To accomplish this, we
analyzed pro-apoptotic markers and observed the influence
of AgNPs in human liver cancer cells (HepG2). As a result
of AgNPs activating the apoptotic machinery, liver cancer
cells were destroyed.
Materials andmethods
Lactobacillus acidophilus isolation, identification,
andmolecular confirmation
Since Lactobacillus acidophilus is a non-toxic, environmen-
tally benign probiotic microbe, it was used in this study after
being isolated from fermented milk products. Two mL of
fermented milk were inoculated with MRS Broth (Thermo
Fisher Scientific, USA) and allowed to access for 24 h at 37
°C, serving as an enrichment average. The enriched samples
were then added to MRS agar and allowed to hatch for 48
h in an anaerobic environment. The colonies were cleansed
by other subcultures that came after their original subcul-
ture. Through the use of primers encoding members of the
facilitator family, which is a large family of membrane trans-
port proteins (F: CAT GTT GGG ATG CAA TGA G, R: TTT
CAA AAC TTG TCC TGC TG), the existence of Lactobacillus
acidophilus was molecularly confirmed [7]. Illustratively,
2 µL of template DNA, 1 µL of each primer (25 µM), and
25 µL of 2 × Taq PCR Master Mix were used in the PCR
reaction. An additional 50 µL of sterilized water was added.
The DNA double helix melts at 95°C during the denaturing
phase. Denaturing, annealing, and elongation cycles come
after this step. Separation of the DNA strands occurs at 95°C
during the denaturing stage. To enable primer binding to
template DNA, the annealing step is performed at 55°C. The
complementary strand of DNA may be synthesized by DNA
polymerase at 72°C thanks to the elongation cycle. Last but
not least, the reaction is terminated at 4°C after the previous
extension step permits any leftover DNA strands to be fin-
ished at 72°C. The isolate's 16S rRNA gene sequences were
compared to reference database sequences in MEGA version
5.0 using the BLAST method [45]. The accession number
for the closest match was found at https:// www. ncbi. nlm. nih.
gov/ nucco re/ OR616 671.1. As a result, the isolates could be
placed in the proper taxonomic position while building the
phylogenetic tree.
The extraction process ofAgNPs
To isolate AgNPs from bacteria, we adapted the method
proposed by Keskin etal. [46]. First, Lactobacillus acido-
philus-derived nano-Ag bacteria were harvested from MRS
broth by centrifugation (1700×g, 15 min, 4ºC). As a result of
three PBS washes and 90 min of incubation at 37ºC, bacteria
were resuspended in NaOH 0.1 M (5 ml of NaOH was used
per 0.1 g of bacteria). After several washes with de-ionized
water (6000×g, 15 min, 4ºC), the lysed bacteria and AgNPs-
clusters were recovered. Sonication was performed on har-
vested clusters containing AgNPs using the UP50H soni-
fier from Hielscher Ultrasonics GmbH (Teltow, Germany).
Finally, vacuum filtration using membrane filters with pore
sizes ranging from 2 to 0.45 mm was used to separate the
dispersed AgNPs from cell debris. AgNPs were stored at 4ºC
as a purified aqueous solution.
Characterization ofAgNPs
Fourier transform infrared spectroscopy (FTIR)
Under the Japanese Alpha e-Bruker spectrometer, AgNPs
were investigated at a resolution of 4 cm−1 and a frequency
of 1000–4000 wave numbers cm–1. Following the acqui-
sition of smoothed spectra, baselines were modified [47].
Comparison with publicly accessible standards was used to
analyze the FTIR spectra.
UV–Vis spectroscopy ofAgNPs
Using a multi-plate reader from Perkin Elmer, we measured
the absorption spectra of purified AgNPs. 96-well plates
were seeded with 100 ml of AgNPs suspended in de-ionized
water per well. Normalized readings were taken against de-
ionized water-filled blank wells [48].

Medical Oncology (2024) 41:106 106 Page 4 of 20
Crystallography andtransmission electron
microscopy (TEM)
XRD diffractograms of AgNPs were obtained using Cu-Kα
radiation (λ = 1.5405 Å), and they were analyzed based on
the Rietveld method in a Bragg–Brentano geometry, and
the structure of the composites was characterized using the
XRD diffractograms [49]. Using TEM (JEOL 2010, Tokyo,
Japan), the AgNPs were examined as per the recommended
procedure [50]. A carbon-coated copper grid was used in
the TEM for mounting PG-AgNPs. Once the sample had
been air-dried at room temperature, its size and shape were
assessed at an acceleration voltage of 200kV.
Determination ofapoptosis
The HepG2 cells that had been given the AgNPs treatment
were collected and given a pH 7.2, 1 × PBS wash [51]. Fol-
lowing that, cells were stained on clean microscope cov-
erslips with acridine orange (AO) (50 µL of 1mg/ml) and
ethidium bromide (EB) (100 µL of 1mg/ml). Cells were
gathered and seen using a fluorescence microscope (ZOE
fluorescent cell imager) after incubation for 20min at 37º C
in the dark (BioRad, USA).
M30 apoptosense assay
The M30 Apoptosense® was used to assess apoptosis lev-
els in HepG2 cells, following the established technique
[52]. The quantification of soluble apoptosis-related and
caspase-cleaved K18 fragments, including the neo-epitope
K18Asp396 in soluble coexpression systems, may be
achieved by the use of the M30 Apoptosense test. The tech-
nique used is a solid-phase sandwich enzyme immunoassay,
in which the samples undergo a reaction with an anti-K18
solid-phase capture antibody. This is followed by the interac-
tion with the HRP (horseradish peroxidase) conjugated M30
antibody, specifically designed to target the K18Asp396 neo-
epitope. The relationship between the absorbance measured
at a wavelength of 450 nm using a Spectrophotometer and
the concentration of the analyte is directly proportional.
Cell culture andtreatment
The culture of HepG2 and WI-38 human lung cells involved
the use of DMEM medium (Lonza, Bio Whittaker®, USA)
(CAS-No. 12614), which was supplemented with 10% fetal
bovine serum (FBS) (Sigma, USA) (CAS-No. 1943609-65-
1). Additionally, the medium was supplemented with 100
mg/ml penicillin and 100 mg/ml streptomycin and main-
tained at a temperature of 37 °C in a humidified incubator
with 5% CO2. The treatments were consistently administered
to cells that were 40–50% confluent after the subculturing of
the cells at an 80%-90% confluency and subsequent splitting
at a ratio of 1:6. All treatments were administered for a dura-
tion of 48 h, with drug dosages equivalent to half of the 50%
inhibitory concentration (IC50) values (IC50/2). To conduct
statistical analysis, three separate cell culture experiments
were carried out, with each experiment being replicated
three times [2, 25].
Cell viability assay
In this study, the culture of HepG2 and WI-38 human lung
cells involved the utilization of DMEM medium (Lonza,
Bio Whittaker®, USA) (CAS-No. 12614). This medium was
supplemented with 10% fetal bovine serum (FBS) (Sigma,
USA) (CAS-No. 1943609-65-1), as well as 100 mg/ml peni-
cillin and 100 mg/ml streptomycin. The cells were main-
tained in a humidified incubator at 37 °C, with the addition
of 5% CO2 to the medium. The treatments were consistently
administered to cells that had reached a confluence level of
40–50%. Prior to treatment, the cells were subcultured when
they got a confluence level of 80%–90% and were divided
at a ratio of 1:6.
The vitality of HepG2 and WI-38 cells was assessed
using an MTT colourimetric test kit obtained from Sigma-
Aldrich, USA. The effects of AgNPs and Sor on cell viabil-
ity were investigated. The cells were grown overnight in
96-well plates at a density of 5 × 103 cells per well after
seeding. Subsequently, the cells were treated with varying
concentrations of AgNPs and Sor (ranging from 0.5 to 100
ng/ml) for a duration of 48 h. A volume of 100 ml of MTT
working solution was added to each well, and the plates
were incubated for a period of four hours at a temperature
of 37°C in the absence of light. Following the removal of the
medium, DMSO was introduced into each well and allowed
to incubate for a duration of five minutes, facilitating the dis-
solution of the purple formazan crystals. The optical density
(OD) at a wavelength of 570 nm was measured using a BIO-
RAD PR4100 microplate reader manufactured in the United
States. To determine the IC50, the vitality of cells subjected
to treatment was compared to that of control cells that were
not treated [26].
Evaluation oftheantioxidant capacity forAgNPs
markers
The glutathione (GSH) levels in HepG2 and WI-38 cell
lysates were evaluated after a 48-h treatment with half of
the IC50 doses of AgNPs and Sor. The quantification of
malondialdehyde (MDA) was conducted in cell lysates of
both HepG2 and WI-38 cells to assess lipid peroxidation
levels [25].

Medical Oncology (2024) 41:106 Page 5 of 20 106
Quantification ofcytokines inAgNPs‑treated cells
supernatants
In HepG2 culture supernatants treated with AgNPs, the
concentrations of IL-33 and TNF-α were assessed. 10,000
cells for HepG2 were planted in 96-well plates. After 24 h
of adhesion, the cells were treated with a 50 µg/ml probiotic
sample for 3 h [53]. ELISA was used to measure the num-
ber of cytokines in the culture supernatants. Results were
revealed as the mean value ± standard deviation (SD) from at
least three replicate tests. The cytokine levels in the culture
supernatants were measured using the sandwich enzyme-
linked immunosorbent assay. According to the manufactur-
er's instructions, a unique ELISA kit (bioscience, San Diego,
CA, USA) for each cytokine was utilized. In an ELISA plate
reader, the optical density of the reaction products was eval-
uated (EnSpire Multimode Plate Reader).
RT‑PCR assessment andRNA extraction
In accordance with the guidelines provided by the manufac-
turer, the extraction of total RNA from cells treated with a
concentration equivalent to half of the IC50 was performed
using the Gene JET RNA extraction kit (Cat#A0156) manu-
factured by Thermo Fisher Scientific. The purity and con-
centration of the extracted RNA were assessed using the
Analytik Jena Scandrop200 instrument from Germany. Sub-
sequently, cDNA synthesis was carried out using the Sen-
siFASTTM cDNA Synthesis Kit (BIO-6505) from Thermo
Co, USA.
Primers were developed for the AMPK, mTOR, MMP-9,
BCL-2, and α-SMA genes in the context of quantitative RT-
PCR. The primer sequences may be found in Supplementary
TableS1. For the PCR reaction, a solution was prepared by
combining 10µL of SYBR green, 2µL of cDNA template,
and 6.4µL of nuclease-free water. The Q5plex detection
device was used to detect rotor gene amplification. After an
initial activation step at a temperature of 95°C for a duration
of 2 min, a series of 45 cycles was conducted. Each cycle
consisted of three steps: a denaturation step at 95°C for 5 s,
an annealing step at 62°C for 10 s, and an extension step at
72°C for 20 s. The study used the comparative 2−ΔΔCt tech-
nique to determine relative expression levels, with the inter-
nal housekeeping gene GAPDH serving as a reference [54].
Molecular docking andADMET
To determine the molecular interactions and scores of
AgNO3-NPs and sorafenib against human MMP-9 and
mTOR, the three-dimensional structures of MMP-9 and
mTOR were retrieved from the RCSB Protein Data Bank
(https:// www. rcsb. org/) database. In addition, the AgNO3
and sorafenib structures were retrieved from PubChem
(https:// pubch em. ncbi. nlm. nih. gov/) database. Furthermore,
the target proteins were prepared by removing water mol-
ecules and attaching ligands using the UCSF Chimera soft-
ware package. The molecular docking interaction of AgNO3
and sorafenib against MMP-9 and mTOR was done using the
AutoDock 4.2 tool and UCSF Chimera while visualized by
BIOVIA Discovery Studio software. ADMET predictions
of AgNO3 and sorafenib were made using the ADMET and
toxicity tools present in BIOVIA Discovery Studio software
[26].
Analyses ofstatistics
Analysis of the data were performed with GraphPad Prism
version 9.0 (GraphPad Software, Inc., La Jolla, CA, USA).
An ANOVA was used to compare data between multiple
groups, followed by Tukey's post-hoc test to compare data
between individual groups. The least significance difference
test was used to determine differences between means in all
tests, with significance defined as P < 0.05 for all tests.
Results
Isolation, 16S rRNA gene sequence analysis,
andbiosynthesis ofAgNPs
To verify identification through molecular analysis, the
strain’s 16S rDNA gene was sequenced. The Lactobacil-
lus acidophilus strain RBIM that has the highest similarity
score to the unknown strain can be identified by applying
the BLAST algorithm to compare a partial fragment of
the strain’s 16S rDNA to a database of known 16S rDNA
sequences. This was further supported by a phylogenetic
tree constructed using the neighbour-joining method, which
revealed that the unknown strain shared 99% similarities
with the Lactobacillus gasseri ATCC-19992, allowing for
identification as Lactobacillus acidophilus strain RBIM
(Fig.1). The sequence has been deposited in GenBank with
accession number #OR616671.
Characterization ofAgNPs
Fourier‑transform infrared spectroscopy (FTIR)
FTIR measurements of dried AgNPs were used to evalu-
ate how biomolecules interact with AgNPs, which may
be responsible for their stability (capping material) in the
medium. During the extraction process, biomolecules can
consist of peptides, proteins, and carbohydrates. A well-
known signature of the infrared region of the electromag-
netic spectrum can be observed in proteins that have amide
linkages between amino acid residues. An FTIR analysis was

Medical Oncology (2024) 41:106 106 Page 6 of 20
conducted to identify possible interactions between silver
salts and protein molecules responsible for the reduction of
Ag + ions and stabilization of AgNPs. The FTIR analysis
of AgNPs was carried out between the wave numbers 1000
cm −1 to 4000 cm −1. The strong peaks at 3750 cm −1, 3300
cm −1, 2800 cm −1, 2600 cm −1, 1650 cm −1, 1450 cm −1, 1290
cm −1, and 1070 cm −1 were observed in the FTIR spectrum
of AgNPs synthesized extracellularly by Lactobacillus aci-
dophilus as shown in (Fig.2). These peaks were attributed
to the presence of silanols, carboxylates, phosphonates, and
siloxanes on the surface of the AgNPs. The presence of these
compounds was an indication of the biogenic synthesis of
the silver nanoparticles.
Illustratively, the peaks observed at wave number 1650
cm −1 are indicative of peptide linkage stretching. In contrast,
1290 cm −1 demonstrated a stretching of CN-amines and a
bending of the NH side of peptides. Peaks in this region can
be attributed to the secondary structure of proteins, hydrogen
bonding between amino acids, and electrostatic interactions
between amino acids. Moreover, these peaks can give infor-
mation about a protein’s flexibility, reactivity, and stability.
The peak at 1070 cm −1 demonstrates that –CH3 structur-
ally bends in amino acids when the chain is stretched. It
is likely that the peaks at 1450 cm–1 are due to changes in
the asymmetric stretch of NO caused by the presence of
nitro compounds. This suggests that the nitro compounds
may be affecting the overall structure of the amino acid,
causing the –CH3 to structurally bend. This could explain
why nitro compounds are responsible for the absorption
spectrum shift. Furthermore, there are two peaks at 2600
cm–1 and 2800 cm–1, which are indicative of CH stretching
in aldehydes. Due to the fact that saturated hydrocarbons
have a –CH saturation vibration, we observed peaks at 3300
cm–1 and 3750 cm–1. This suggests that the aldehydes were
more complex than the saturated hydrocarbons since they
had additional CH stretching vibrations. The additional
CH stretching vibrations in the aldehydes are necessary to
accommodate the double bond, which requires more energy
than the single bond in saturated hydrocarbons. This is
why the nitro compounds are responsible for the shift in
absorption spectra and why the CH stretching vibrations
for the aldehydes are more complex than those in saturated
hydrocarbons.
UV–Vis spectroscopy ofAgNPs
As part of a study on the derivation and stability of silver
oxide nanoparticles in aqueous colloidal solutions, UV–vis-
ible spectral analysis was used to study their synthesis and
stability. As can be seen from (Fig.3), a strong peak has
been observed at 425 nm. This peak was indicative of the
formation and stability of silver oxide nanoparticles, provid-
ing further insight into the study. In general, the intensity
increase could be interpreted as a result of a greater number
of nanoparticles that are formed as a consequence of reduc-
ing silver nitrate that is present in aqueous solutions as a
result of the reduction process.
XRD andTEM analysis
The phase formation and purity of the nanoparticles could
be ascertained by utilizing XRD measurement. Based on
the XRD analysis (Fig.4a), it can be inferred that AgNPs
are crystalline due to their narrow and sharp peaks. Further-
more, the XRD analysis also indicates that AgNPs are of
high purity, as the peaks maintain their sharpness and inten-
sity throughout the spectrum. A face-centred cubic structure
is present in the silver nanocrystal, as indicated by intense
peaks observed at 38° in the 2θ range. The intensity of the
Fig. 1 Analysis of the 16S rDNA of Lactobacillus acidophilus strain
RBIM phylogenetic tree available in GenBank. The tree suggests that
the strain RBIM belongs to the same species as other Lactobacillus
acidophilus strains. Furthermore, the phylogenetic tree indicates that
the strain RBIM is distinct from other Lactobacillus species

Medical Oncology (2024) 41:106 Page 7 of 20 106
peaks also indicates that the particles are small and uniform
in size, with a narrow size distribution. The shape of the
peaks is also indicative of the crystallinity of the particles,
as sharp peaks indicate that the particles are crystalline and
uniform in size and shape. This corresponds to the silver
file number 04-0783 on the Joint Committee on Powder
Diffraction Standards (JCPDS) standard powder diffrac-
tion card. The TEM micrographs revealed a homogeneous
size distribution of the AgNPs, suggesting that the particles
were not aggregated and could retain their original shape
(Fig.4b). The particles' estimated sizes ranged from 17 to
19 nm, which is within the typical range for AgNPs. Addi-
tionally, the spherical form and good dispersion of AgNPs
were revealed by the TEM micrograph. This indicates that
the AgNPs are stable and appropriate for usage in a variety
of applications as they are uniformly dispersed and of the
right size and shape.
Effect ofAgNPs onapoptosis
In the treatment with AgNPs, the cells went through an
apoptosis process, which was visualized by staining the cells
with AO/EB. Observations of the fluorescence of normal
viable control cells at the microscopic level revealed that
the color of the cells was green due to the diffusion of an
AO compound into the cell membrane. Apoptotic cells were,
on the other hand, observed to have orange-coloured bodies
due to the shrinkage and blebbing of the cells caused by the
AgNP treatment compared to non-apoptotic cells. Apoptotic
cells are typically characterized by yellow-green deposits of
AO staining on the nuclei of early-stage apoptotic cells. As
the apoptotic stage progressed, EB staining on the nuclei
of late-stage apoptotic cells became more concentrated and
asymmetrically localized. In necrotizing cells, the volume
of the cells increased, and the fluorescence at the periph-
ery of the cells was uneven and orange-red. There has been
Fig. 2 An FTIR spectrum of Lactobacillus acidophilus strain RBIM displays peaks at different wave numbers. Lactobacillus acidophilus strain
RBIM exhibited various bands characteristic of AgNPs, which indicates that the AgNPs were highly effective at this specific frequency
0100 200300 400500 600 70
08
0
0
0.3
0.8
1.3
1.8
2.3
2.8
3.3
3.8
Wavelength(nm
Ab
sor
b
ance
Fig. 3 Analysis of AgNPs extractions by UV–Vis from lysed Lacto-
bacillus acidophilus strain RBIM cultured in AgNO3 and dispersed
in deionized water. The peak is located at 575–585 nm. In this peak,
AgNPs were extracted from the solution, indicating that the synthe-
sized AgNPs were successfully extracted

Medical Oncology (2024) 41:106 106 Page 8 of 20
a suggestion that apoptosis is caused by AgNPs that are
involved in disrupting the mitochondrial respiratory chain,
resulting in reactive oxygen species (ROS) being produced
and damaging nucleic acids. The uneven localization of EB
staining in apoptotic cells suggests that the mitochondria
are undergoing rapid and abnormal fragmentation, leading
to the release of EB staining into the cytosol. In necrotiz-
ing cells, the ROS produced by the AgNPs further damages
the mitochondria, leading to further fragmentation and the
release of EB staining from the cells (Fig.5a).
Induction ofapoptosis inHepG2 treated withAgNPs
The concentrations of the M30 epitope in HepG2 cell
extracts (Fig.5b), which serve as an indicator of caspases
3/7/9 activation and subsequently apoptosis, were seen to be
Fig. 4 A AgNPs XRD spectrum
recorded for Lactobacillus
acidophilus. The spectrum
presented peaks at 2θ values
of 38.2°. These peaks are
consistent with the crystalline
structure of the AgNPs. This
indicates that the AgNPs were
successfully synthesized and
interacted with the bacteria. B
AgNPs micrographed by TEM.
Approximately 17 nm in diam-
eter with a standard deviation of
2 nm were the mean sizes of the
AgNPs. Throughout the sample,
the particles were uniformly
distributed and spherical. TEM
micrographs revealed that
AgNPs were uniformly sized
and distributed
Fig. 5 A AO/EB fluorescence
images for (Ab) Control and
(Ab),(AC) 50 μg/ml AgNPs-
treated-HepG-2 cells. The
arrows indicate that the cells are
experiencing apoptosis, which
results in chromatin condensa-
tion and fragmentation. Early-
stage apoptosis is characterized
by chromatin condensation,
whereas late-stage apoptosis
is characterised by chromatin
fragmentation. B The amount
of M30 in cell extracts. HepG2
cells were given an AgNPs
treatment of 50 µg/ml over
24–48 h. The M30 epitope is
also detected in HepG2 cells,
indicating cell death. A higher
level of these molecules was
found in AgNPs-treated cells
compared to controls, reach-
ing a peak after 24 h. The
graphs reveal the results as the
mean ± SD of three replicates

Medical Oncology (2024) 41:106 Page 9 of 20 106
elevated in cells treated with AgNPs in comparison to the
control group. The highest levels were observed after 23 h
of treatment. This indicates that AgNPs can trigger apop-
tosis in HepG2 cells, potentially due to the oxidative stress
caused by the AgNPs. The oxidative stress can activate the
caspases, which in turn triggers the M30 epitope production
and apoptosis. This suggests that AgNPs may be an effective
therapeutic agent for treating certain cancers, as they can
lead to apoptosis of the cancer cells. Additionally, AgNPs
can be used in combination with other therapies, such as
chemotherapy, to further increase their efficacy.
Cytotoxicity evaluation
MTT tests were carried out against HepG2 and WI-38 cells
to evaluate AgNPs’ cytotoxic potential. A range of 1.56–100
µg/ml of AgNPs was used to treat cells for 48 h. Both cell
lines showed dose-dependent reductions in cell viability
after 24 h of exposure to AgNPs. Assays with HepG2 and
WI-38 demonstrated that AgNPs inhibited cell growth (IC50
4.217 µg/ml and 154.1 µg/ml, respectively) (Fig.6). HepG2
and WI-38 cells, however, revealed reduced proliferation
after 48 h post-AgNPs treatment (IC50 = 119.5 µg/ml and
8.98 µg/ml, respectively). This implies that AgNPs were
more effective in reducing the growth of HepG2 cells than
WI-38 cells after 48 h. This could be due to the fact that
HepG2 cells are more susceptible than WI-38 cells to the
effects of AgNPs. Additionally, the decrease in cell viability
could be caused by the AgNPs binding to the cell membrane
and interfering with the cell cycle.
AgNPs treatment reduced MDA intheHepG2 cell
line
In the HepG2 and WI-38 cell lines, it was shown that cells
treated with AgNPs exhibited elevated levels of GSH com-
pared to both the untreated control cells and the cells treated
with Sor. As a result, the AgNPs therapy contributed to the
most significant reduction of MDA levels in HepG2 cells as
well as WI-38 cells when compared to a control solution.
However, the MDA levels of HepG2 and WI-38 cells that
were treated with Sor were significantly higher than those of
the control cells (Fig.7). This suggests that AgNPs may be a
more effective therapy than Sor for reducing the MDA levels
of HepG2 and WI-38 cells. Additionally, the GSH levels in
the AgNPs-treated cells may have been increased to provide
a defence against oxidative damage induced by the MDA.
Influence ofAgNPs‑treated HepG2 Cells
onthesecretion ofpro‑inflammatory cytokines
AgNPs cause an increase in the production of inflammation-
related cytokines, such as TNF-α and IL-33, in HepG2 cells
(Fig.8). Immune response is enhanced by these cytokines,
which are part of the body's natural response to infections
and injuries. This stimulation is mediated by the activa-
tion of the AMPK pathway. The AMPK pathway is a cru-
cial regulator of inflammation, and its activation leads to
inflammatory cytokines. Therefore, the AMPK pathway is
an important signaling component in the response to AgNPs.
Effect ofAgNPs onsignalling pathways
andapoptosis‑autophagy‑related marker genes
The study used reverse transcription polymerase chain reac-
tion (RT-PCR) assays to figure out the effects of AgNPs
and Sorafenib (Sor) on the expression levels of AMPK and
mTOR. Additionally, the study investigated the influence of
these substances on several markers associated with angio-
genesis, metastasis, apoptosis, and autophagy, including
MMP-9, BCL-2, and alpha-smooth muscle actin (α-SMA).
As a comparison to the cell control, AgNPs treatment in
WI-38 cells caused upregulation of AMPK and downregu-
lation of mTOR, MMP-9, BCL-2, and α-SMA, whereas, in
control cell culture, AgNPs treatment did not show a sig-
nificant effect in BCL-2 compared to Sor (Fig.9). These
results revealed that AgNPs treatment had an impact on the
expression of these genes, which suggests that AgNPs have
Fig. 6 Growth inhibition curves
in A HepG2 cells treated with
AgNPs; B WI-38 cells treated
with AgNPs. The MTT assay
confirmed that the AgNPs
generated a dose-dependent
inhibition in cell proliferation
in both cell lines, with more
significant inhibitory activity in
the HepG2 cells. Moreover, the
inhibition was also prominent in
HepG2 cells after 48 h of treat-
ment. This implies that AgNPs
may have a long-lasting effect
on HepG2 cells

Medical Oncology (2024) 41:106 106 Page 10 of 20
Fig. 7 Oxidative stress mark-
ers and antioxidant activity
of AgNPs and Sor A GSH of
AgNPs in both HepG2 and
WI-38 cell lines; B GSH of Sor
in both HepG2 and WI-38 cell
lines; C MDA concentration in
control and treated HepG2 and
WI-38 cells with AgNPs; D
MDA concentration in control
and treated HepG2 and WI-38
cells with Sor. *(P ≤ 0.05),
**(P ≤ 0.01) and ***(P ≤ 0.001)
means significantly different
from control. The data were
analyzed in triplicate and
expressed as mean ± SD
Fig. 8 AgNPs induce pro-inflammatory cytokines in liver cancer
cells. Following treatment with AgNPs, Liver cancer cells produce
more cytokines that promote inflammation. A The production of
TNF-α by AgNPs-treated HepG2 cells; B The production of IL-33 by
AgNPs-treated HepG2 cells. AgNPs concentration of 50 µg/mL was
added to the cells for 3 h. ELISA was used to determine the cytokine
concentrations in the culture supernatants. The findings are the
mean ± SD (P < 0.05, Student’s t-test) of three replicates

Medical Oncology (2024) 41:106 Page 11 of 20 106
the potential to regulate the expression of genes involved in
cell metabolism, proliferation, and apoptosis.
The treatment of HepG2 cells with AgNPs resulted in
the most significant upregulation of AMPK compared to
Sor, as revealed in (Fig.10). A comparison of the expres-
sion of mTOR, MMP-9, BCL-2, and mTOR in HepG2 cells
treated with AgNPs and the control was found to be the
most strongly suppressed, compared to Sor and control. This
suggests that AgNPs may have the ability to modulate the
AMPK/mTOR signalling pathway, which can lead to the
suppression of MMP-9, BCL-2, and α-SMA expressions, as
well as an increase in apoptosis and autophagy. This modu-
lation of the AMPK/mTOR signalling pathway by AgNPs
could be responsible for the observed decrease in cell via-
bility, decreased proliferation, and increased apoptosis and
autophagy seen in HepG2 cells treated with AgNPs.
Molecular docking andADMET assessment
The docking interactions of AgNO3 and sorafenib with
MMP-9 and mTOR are revealed in Table1 and (Figs.11,
12), respectively. The compound AgNO3 exhibited
interactions with the binding sites of MMP-9 and mTOR,
resulting in binding energies of −3.1 and −3.7 kcal/mol,
respectively. Sorafenib had a binding affinity of −7.5 kcal/
mol and −9.1 kcal/mol to the MMP-9 and mTOR inter-
action sites, respectively. The observed energies suggest a
significant affinity between AgNO3 and the binding sites of
MMP-9 and mTOR, as well as between sorafenib and the
binding sites of MMP-9 and mTOR. This suggests that the
compounds AgNO3 and sorafenib possess the capability to
form complexes with MMP-9 and mTOR, hence possibly
impeding their respective functionalities.
The hepatotoxicity of sorafenib and the mutagenic likeli-
hood of AgNO3 were predicted using ADMET and Ames’
methodologies, respectively. The aforementioned predic-
tions underscore the need for further assessment of the
hepatotoxicity and mutagenicity of AgNO3. The results
obtained from the ADMET and Ames' predictions indi-
cated that the hepatotoxicity of sorafenib and mutagenicity
of AgNO3 exceeded initial expectations, therefore suggest-
ing that these substances cannot be disregarded as possible
dangers (Table2). This observation underscored the need
for conducting more comprehensive investigations on the
Fig. 9 Effect of AgNPs or Sor treatment on the relative expression
of apoptosis-autophagy-related genes on WI-38 normal cell lines
A AMPK gene-fold change; B mTOR gene-fold change; C MMP-9
gene-fold change obtained by RT-PCR; D BCL2 gene-fold change;
E α-SMA gene-fold change that obtained by qRT-PCR. The graphs
of genes are plotted as 2−∆∆Ct-fold changes. Data are expressed as
mean ± SEM. A one-way ANOVA comparison test was employed
to determine the significant differences (P < 0.05). *(P ≤ 0.05),
**(P ≤ 0.01), ***(P ≤ 0.001), ns, (non-significant, P ≥ 0.05)

Medical Oncology (2024) 41:106 106 Page 12 of 20
hepatotoxic and mutagenic effects of AgNO3 to further our
comprehension of the possible hazards it may provide.
Discussion
Considerable effort has been put into creating effective
anti-cancer therapies in recent decades [55]. Nanomateri-
als of all types, including AgNPs, are considered [56].
AgNPs depend on how the targeted cell behaves biologi-
cally. Thus, they may enhance standard treatments through
the induction of oxidative stress-mediated autophagy and/
or apoptosis [57, 58]. In developing novel, effective thera-
peutic tools, the current study has focused on the pos-
sibility of using AgNPs in oncotherapy. To characterize
realistic opportunities for using AgNPs to treat cancer,
AgNPs have been investigated for their ability to induce
apoptosis in liver cancer cells as well as their ability to
play a role in the immune response to these cells. To reach
this goal, we examined signalling pathways in HepG2 cells
and gene expression profiles associated with apoptosis and
autophagy. Furthermore, we sought to identify potential
mechanisms by which these signalling pathways might be
regulated.
In synthesizing AgNPs, the green biosynthesis and sta-
bilization process by Lactobacillus acidophilus plays a key
role. This is because it is a relatively cheap and safe process,
as well as because it produces high-quality nanoparticles
[59]. In synthesizing silver nanoparticles, the green biosyn-
thesis and stabilization process by Lactobacillus acidophilus
plays a key role. This is because it is a relatively cheap and
safe process, as well as because it produces high-quality nan-
oparticles. Reducing aqueous silver ions to AgNPs caused
the color change in the Lactobacillus acidophilus superna-
tant. The reduction process is caused by the bacteria’s ability
to produce enzymes that catalyze the reduction of silver ions
[12]. This process effectively produces high-quality nano-
particles because it ensures that the nanoparticles are of a
consistent size and shape, which is vital for many appli-
cations [60]. Additionally, the Lactobacillus acidophilus
supernatant helps to reduce the toxicity often associated with
silver nanoparticles [61].
Fig. 10 Effect of AgNPs or Sor treatment on the relative expression
of apoptosis-autophagy-related genes on HepG2 cell lines A AMPK
gene-fold change; B mTOR gene-fold change; C MMP-9 gene-fold
change obtained by RT-PCR; D BCL2 gene-fold change; E α-SMA
gene-fold change that obtained by qRT-PCR. The graphs of genes are
plotted as 2−∆∆Ct-fold changes. Data are expressed as mean ± SEM.
A one-way ANOVA comparison test was employed to determine
the significant differences (P < 0.05). *(P ≤ 0.05), **(P ≤ 0.01),
***(P ≤ 0.001), ns, (non-significant, P ≥ 0.05)

Medical Oncology (2024) 41:106 Page 13 of 20 106
The FTIR method detects bond characteristics based
on vibrations, identifying functional groups and revealing
information regarding covalent bonds between them [62].
A significant feature of marine bacteria is that they possess
functional groups, making them a potential biotechnologi-
cal resource [63]. Our FTIR spectra indicate that the AgNPs
exhibit these peaks, which were attributed to silanols, car-
boxylates, phosphonates, and siloxanes on the surface of
the silver nanoparticles. These functional groups, such as
carboxylates and phosphonates, are essential for bacteria
to survive in the ocean environment [64]. The presence
of siloxanes on the surface of the silver nanoparticles also
makes them an attractive option for biotechnological appli-
cations, as these molecules can bind with other molecules,
making them useful in various chemical reactions [65].
Additionally, AgNPs were more stable in a solution with a
high CN-amine concentration [66]. This stability is caused
by the electrostatic interactions between the AgNPs and CN-
amines, which causes the AgNPs to stretch [67]. The bend-
ing of the NH side of peptides is caused by the interactions
between the AgNPs and peptides, which causes the AgNPs
to bend [68].
It is evident from our data that the surface plasmon
resonance peak bands in the UV–Vis spectrum are centred
around 425 nm, which is a characteristic feature of AgNPs. It
was observed that the absorption peak at 425 nm decreased
in width, and the peak intensity increased as a result. As
Sankar etal. [60] suggested, the peak intensity may indicate
the presence of smaller, spherical nanoparticles with some
agglomeration. Our study’s results also agree with those of
previously published studies [69, 70].
Our data revealed that AgNPs treatment leads the cells
to apoptosis, which was visualized using AO/EB staining
of the cells. This could be explained by the fact that AgNPs
treatment caused a decrease in the mitochondrial mem-
brane potential, leading to an increase in ROS, which in
turn causes cells to die, a process known as apoptosis [71].
Furthermore, AgNPs treatment also induced the expression
of apoptotic proteins, such as Bax and caspase-9 [72]. Addi-
tionally, Barabadi etal. [73] revealed that induced overpro-
duction of ROS and interruption of ATP synthesis by AgNPs
prevent the mitochondrial respiratory chain from functioning
properly, resulting in mitochondrial pathway apoptosis. The
results of their works provide strong preliminary evidence
that biogenic AgNPs are effective against hepatic cancer
cells in invitro studies, which are involved in the apoptotic
process[74]. These data were confirmed by the presence of
the biomolecular layer on the surface of AgNPs, which is
expected to play an essential role in the apoptosis induced
by AgNPs in liver cancer cells [75], as described here. The
present study demonstrated that 50 µg/ml AgNPs inhibited
HepG2 cancer cells' growth. Significantly, HepG2 liver can-
cer cells responded to AgNPs by inducing intrinsic apoptotic
mechanisms. As a result of interactions between AgNPs'
biomolecular layer and proteins on HepG2 cancer cells, a
cascade of events that lead to apoptosis may unfold. At these
events, caspases, which are the main executors of apoptosis,
become activated. Additionally, other proteins are activated,
Table 1 Molecular interactions
of AgNPs and sorafenib against
matrix metalloproteinase-9
(MMP-9) and mammalian target
of rapamycin (mTOR)
Drugs Targets Docking scores
(kcal/mol) Interaction residues Interaction type
AgNPs MMP-9 −3.1 CYS99 Conventional hydrogen bond
HIS401 Charge-charge
GLU402 Charge-charge
mTOR −3.7 PHE2314 Charge-charge
ARG2317 Conventional hydrogen bond
THR2318 Conventional hydrogen bond
THR2321 Conventional hydrogen bond
GLU2388 Charge-charge
ASN2395 Conventional hydrogen bond
Sorafenib MMP-9 −7.5 ARG51 Conventional hydrogen bond
ARG95 Hydrophobic
ASP182 Hydrogen bond
ASP182 Charge-charge
mTOR −9.1 ILE1939 Hydrophobic
PRO1940 Conventional hydrogen bond
PRO1940 Halogen
PRO1975 Hydrophobic
GLU2196 Conventional hydrogen bond
MET2199 Conventional hydrogen bond

Medical Oncology (2024) 41:106 106 Page 14 of 20
such as p53, which regulates apoptosis and cell cycle. There
have been numerous reports describing AgNPs as an inhibi-
tor of liver cancer cell growth both invitro and invivo [73,
76]. Furthermore,
In cancer treatment, the MTT assay can assess the cyto-
toxicity of substances by determining the number of viable
cells. According to the MTT results, AgNPs were toxic
to HepG2 cells at a 50 μg/ml concentration. Additionally,
AgNPs showed a high ability to inhibit cell growth, with
the most outstanding inhibition occurring at 100 μg/ml. This
indicates that AgNPs may be a potential cancer treatment. In
similar research, other researchers have documented AgNPs'
cytotoxicity in several cancer cells, including oral cancer cell
lines, breast cancer, and human cervical cancer [77]. This
could be attributed to AgNPs causing oxidative stress and
membrane damage in cells, which can lead to cell death.
Furthermore, AgNPs can cause apoptosis, a programmed
cell death process, in cancer cells and could potentially kill
cancer cells, making them a potential cancer treatment [78].
The AgNPs treatment significantly augmented the con-
centration of GSH in these cells, neutralizing both MDA
and GSH levels. A single Sor was added to both HepG2 and
WI-38 cells. However, when HepG2 and WI-38 cells were
coated with AgNPs, MDA levels decreased significantly,
while those treated with Sor increased. This suggests that
AgNPs have an antioxidant effect, while Sor has an antioxi-
dant and pro-oxidant effect. The antioxidant effect of AgNPs
is likely due to its ability to bind to free radicals and reduce
their activity, while the pro-oxidant effect of Sor is likely
due to its ability to oxidize and damage proteins, lipids, and
nucleic acids [79].
A further observation demonstrating AgNPs’ apoptosis-
inducing ability was IL-33 and TNF-α secretion by cells
treated with AgNPs. Cancerous cells have been reported
Fig. 11 The docking interactions of AgNO3 with A MMP-9 and B mTOR. MMP-9 and mTOR binding sites were bound by the compound
AgNO3, leading to binding energies of −3.1 and −3.7 kcal/mol, respectively

Medical Oncology (2024) 41:106 Page 15 of 20 106
Fig. 12 The docking interactions of sorafenib with A MMP-9 and B mTOR. The binding affinities of sorafenib to the MMP-9 and mTOR inter-
action sites were −7.1 kcal/mol and −9.5 kcal/mol, respectively
Table 2 ADMET and Ames
predictions of AgNO3 and
sorafenib
ADME prediction Ames’ prediction
Drugs Solubility
level BBB level Excretion
(CYP2D6) Hepatotoxicity PPB
AgNO34 3 False True False Mutagen
Sorafenib 1 4 False True True Non-Mutagen

Medical Oncology (2024) 41:106 106 Page 16 of 20
to secrete pro-inflammatory cytokines, which regulate host
immune responses [57]. IL-33 and TNF-α are pivotal in
regulating inflammation and immune responses [80]. The
release of these cytokines by AgNPs is thought to cause the
observed apoptosis-inducing ability [81]. This suggests that
the AgNPs may be able to activate the apoptosis pathway
in cancer cells and induce the activation of other immune
cells, such as dendritic cells and natural killer cells, which
may lead to a more effective immune response [82, 83]. It
is worth noting that the cytokines IL-33 and TNF-α, which
were observed in the supernatants of the cells treated with
AgNPs, have previously been documented as being produced
by cancer cells undergoing apoptosis and playing a role in
the signalling of dendritic and natural killer cells [84, 85].
One of the well-known characteristics of cancer cells is
their ability to avoid apoptosis. Furthermore, autophagy
suppresses tumorigenesis in cancer cells by inhibiting their
survival and inducing their death [86]. Therefore, we ana-
lyzed intrinsic and extrinsic genes associated with apoptosis
and autophagy in our study. AgNPs significantly upregulated
AMPK expression, while AgNPs significantly suppressed
mTOR, MMP-9, BCL-2 and α-SMA expression. Our results
provide evidence that AgNPs have the potential to regulate
apoptosis and autophagy in cancer cells, which could be
a promising therapeutic approach for cancer treatment. As
mTOR/BCL-2 regulates the apoptotic pathway, this study
may be supported by its classification as an anti-apoptotic
protein. The results here reveal that AgNPs cause the HepG2
cell line to enter apoptosis by downregulating BCL-2. Sev-
eral studies have linked AgNPs to apoptosis by lowering
mTOR/BCL-2 in cancerous cells [87, 88]. However, apop-
tosis can be sustained depending on the balance between cell
division and cell death.
Novel targeted therapies capable of inducing apoptosis
or enhancing cancer cells’ susceptibility to established cyto-
toxic medicines have been successfully created. This can be
attributed to the increasing ubiquity of intrinsic and extrinsic
apoptotic triggering mechanisms. AgNPs induce apoptosis,
inhibiting the AMPK/mTOR pathway [37]. This study indi-
cates that the AMPK signalling pathway was effectively
repressed in HepG2 cells. In contrast, the translocation of
mTOR and BCL-2 was notably diminished in cells treated
with AgNPs. These results imply that AMPK inhibition may
be implicated in apoptosis. The observed behaviour can be
attributed to AMPK's crucial role as a metabolic sensor
and its involvement in regulating cell growth. Furthermore,
AMPK negatively regulates the mTOR signalling pathway,
inhibiting cancer proliferation and growth [88]. Integrating
signals from the PI3K/Akt pathway, AMPK also governs
cell survival, proliferation, and angiogenesis [89].
Moreover, it is worth noting that HCC commonly exhibits
elevated mTOR levels associated with early recurrence and
a poor prognosis [25, 90]. Consequently, targeting mTOR
inactivation has been proposed as a potential therapeutic
approach to limiting cancer cell growth in HCC. Thus, the
obtained findings align with our theoretical framework, sug-
gesting that AgNPs trigger autophagy via AMPK/mTOR/
BCL-2 signalling pathway and that autophagy plays a piv-
otal role in AgNPs-induced cellular collapse.
Molecular docking analysis has confirmed the antiprolif-
erative and apoptotic effects of AgNPs on HepG2 cells. This
study has found the interaction between AgNO3 and Sor and
their potential targets, including MMP-9 and mTOR. The
genes under investigation have considerable significance in
converging apoptotic and proliferation pathways. The com-
putational analysis results demonstrated that using AgNPs
may efficiently suppress the activity of MMP-9 and mTOR.
The observed suppression led to a decrease in the expression
of MMP-9, which ultimately resulted in a reduction of the
expression of mTOR. The inhibition of the MMP-9-BCL-
2-dependent AMPK activation pathway by AgNPs leads to
the suppression of mTOR activation, hindering the subse-
quent pathways linked to cancer proliferation.
Moreover, via the mechanism of AMPK downregula-
tion, AgNPs significantly diminish the intracellular levels
of AMPK, decreasing the abundance of mTOR available
to initiate downstream signalling cascades. This could be
explained by the fact that AMPK is a crucial regulator of
mTOR, and its downregulation by AgNPs leads to a decrease
in the activity of mTOR, which would otherwise initiate a
cascade of signalling events that would lead to the activation
of the pathways associated with cell proliferation and dif-
ferentiation [91]. For instance, the AMPK/mTOR pathway
is essential for the translation of proteins involved in cell
growth and proliferation, and a decrease in the abundance
of AMPK can thus inhibit cell growth [92].
Conclusion
This study shows that AgNPs exhibit an inhibitory effect
on cell proliferation and induce apoptosis in hepatocellular
carcinoma HepG2 cells. The AMPK/mTOR inhibitory sig-
nalling pathway plays a substantial role in developing HCC.
The present study elucidates the potential apoptotic anti-
HCC characteristics of AgNPs by suppressing the AMPK/
mTOR signalling pathway. The cytotoxic action of AgNPs
was observed to hinder the proliferation of HepG2 cells,
with an IC50 of 5.21 µg/ml. In the research on apoptotic
activity, it was observed that the administration of AgNPs
resulted in a considerable induction of apoptotic cell death in
HCC cells. This effect was observed following a 48-h treat-
ment with AgNPs, which led to the release of pro-inflam-
matory cytokines. In addition, the highest levels of mTOR,
MMP-9, BCL-2, and α-SMA inhibition coincide with the
upregulation of AMPK expression in HepG2 cells treated

Medical Oncology (2024) 41:106 Page 17 of 20 106
with AgNPs. This study investigated the AMPK/mTOR sig-
nalling pathway of AgNPs through integrated methodologies
both invitro and in silico. The cumulative findings of our
research provide more evidence in favour of the potential
anti-cancer efficacy of AgNPs as a viable alternative therapy
strategy for HCC. Nevertheless, it is important to carry out
further invivo research using animal models to authenti-
cate these results. AgNPs can selectively target tumor cells
owing to their distinct structure and composition. Moreover,
they can selectively attach to certain receptors located on the
neoplastic cells, augmenting their efficacy in inducing cell
death. Additionally, the AgNPs exhibit immunostimulatory
properties, facilitating further suppression of tumor cells.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s12032- 024- 02330-8.
Acknowledgements NA
Author contributions Conceptualization, AE, TE, MOA, AHE: For-
mal analysis, AE, NANA, MGH, TE, NFI, JH: Investigation, AE, TE,
and MGH: Project administration, AHE, NFI, JH, GSG, MMA: Soft-
ware, NKA, ISA, AE, NANA: Validation, AE, TE: Visualization, AE,
TE, MGH: Writing—original draft, AE:Writing—review and editing,
AE. All authors have read and agreed to the published version of the
manuscript.
Funding Open access funding provided by The Science, Technology &
Innovation Funding Authority (STDF) in cooperation with The Egyp-
tian Knowledge Bank (EKB). NA.
Data availability The datasets generated and/or analyzed during the
current study are available in the GeneBank repository with accession
number OR616671.
Declarations
Competing interest The authors declare no competing interests.
Ethical approval and Consent to participate NA
Consent for publication NA
Open Access This article is licensed under a Creative Commons Attri-
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need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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